Branched polymers and their conjugates

ABSTRACT

The present invention is directed to branched reactive water-soluble polymers comprising at least two polymer arms, such as poly(ethylene glycol), attached to a central aliphatic hydrocarbon core molecule through heteroatom linkages. The branched polymers bear at least one functional group for reacting with a biologically active agent to form a biologically active conjugate. The functional group of the branched polymer can be directly attached to the aliphatic hydrocarbon core or via an intervening linkage, such as a heteroatom, -alkylene-, —O-alkylene-O—, -alkylene-O-alkylene-, -aryl-O—, —O-aryl-, (—O-alkylene-) m , or (-alkylene-O—) m  linkage, wherein m is 1-10.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/290,082, filed Nov. 7, 2002, issued as U.S. Pat. No. 7,026,440, whichclaims the benefit of priority of Provisional Application Ser. No.60/337,613, filed Nov. 7, 2001, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to branched, reactive water soluble polymersuseful for conjugating to biologically active molecules and to methodsfor making and utilizing such polymers.

BACKGROUND OF THE INVENTION

Covalent attachment of the hydrophilic polymer poly(ethylene glycol),abbreviated PEG, is a highly advantageous method of increasing watersolubility and bioavailability and extending the circulation time ofmany biologically active molecules, particularly hydrophobic molecules.For example, it has been shown that the water-insoluble drug,paclitaxel, when coupled to PEG, becomes water-soluble. Greenwald, etal., J. Org. Chem., 60:331-336 (1995). The total molecular weight of thepolymer or polymers attached to the biologically active molecule must besufficiently high to impart the advantageous characteristics typicallyassociated with PEG polymer attachment, such as increased watersolubility and circulating half life, while not adversely impacting thebioactivity of the parent molecule.

Proteins and other molecules often have a limited number of reactivesites available for polymer attachment. Often, the sites most suitablefor modification via polymer attachment play a significant role inreceptor binding, and are necessary for retention of the biologicalactivity of the molecule. As a result, indiscriminate attachment ofpolymer chains to such reactive sites on a biologically active moleculeoften leads to a significant reduction or even total loss of biologicalactivity of the polymer-modified molecule. To form conjugates havingsufficient polymer molecular weight for imparting the desired advantagesto a target molecule, prior art approaches have typically involvedeither (i) random attachment of numerous polymer arms to the molecule,thereby increasing the risk of a reduction or even total loss inbioactivity of the parent molecule, or (ii) attachment of one or twovery long polymer chains. Unfortunately, the use of very high molecularweight linear polymer chains is problematic because of the difficultyand expense associated with their preparation, purification, andassociated instability.

Branched polymers comprising a plurality of polymer arms attached to acentral core and having a single reactive group for conjugation to abiologically active molecule have been described in U.S. Pat. Nos.5,643,575 and 5,932,462. Both patents describe branched polymers formedby covalent attachment of a water-soluble polymer such as an end-cappedPEG to a central core molecule bearing amino groups, such as lysine or1,3-diamino-2-propanol. Although these branched polymers are useful forattaching a high molecular weight polymer to a molecule at a singleattachment site without using an extremely long polymer chain, themethods of forming the branched PEG molecules of the prior art isdifficult and requires extensive purification of the PEG polymers priorto attachment to the core molecule and also purification/removal ofpartially pegylated polymer intermediates.

There remains a need in the art for new branched polymer reagents thatprovide the benefits associated with branched polymers (i.e., highoverall molecular weight in a single non-linear polymer molecule), butare easier to synthesize or provide more flexibility in their designthan prior art reagents.

SUMMARY OF THE INVENTION

The present invention is based upon the development of branched,reactive water-soluble polymers useful for conjugation to biologicallyactive molecules in a manner that tends to avoid a significant reductionin the biological activity of the molecule while still providing thebenefits of water-soluble polymer conjugation. The branched polymers ofthe invention can be readily synthesized from a number of aliphatic corestructures that do not require the presence of activating groupssuitable for coupling to an activated linear polymer, such assuccinimidyl carbonate end-capped poly(ethylene glycol) or the like, forbuilding the branched water soluble polymer. That is to say, thepreparation of the polymers of the invention is not hampered by the needto utilize core structures having reactive functional groups necessaryfor coupling with the polymer arms, since the polymer portions of themolecule are generally built directly onto the core by polymerization ofsuitable monomer units.

In one aspect, the present invention provides a branched, reactivewater-soluble polymer comprising at least two polymer arms, such aspoly(ethylene glycol), attached to a central core molecule throughheteroatom linkages such as ether linkages. The central core molecule isan aliphatic hydrocarbon having a length of at least three carbon atoms.The branched polymers of the invention are preferably although notnecessarily monofunctional (i.e., having one reactive functional groupsuitable for covalent attachment to a biologically active agent), andthe single functional group is preferably attached, optionally via anintervening linkage, to the aliphatic hydrocarbon core molecule.

Suitable polymers for use in preparing the branched polymer structuresof the invention include poly(alkylene glycols), poly(oxyethylatedpolyol), poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol),polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), poly(acrylicacid), carboxymethyl cellulose, hyaluronic acid, hydroxypropylmethylcellulose, and copolymers, terpolymers, and mixtures thereof. In oneembodiment of the invention, the polymer is a poly(ethylene glycol).

In another aspect, the invention provides a biologically activeconjugate comprising a biologically active molecule, such as a protein,covalently attached to a branched polymer as described above. Thebiologically active molecule is preferably attached to the branchedpolymer via a linkage formed by reaction of a reactive functional groupon the branched polymer with a suitable functional group on thebiologically active molecule.

In yet another aspect, the invention provides a method of preparingbranched reactive polymers comprising poly(alkylene glycol) polymerarms. The method includes polymerization of alkylene oxide monomerunits, such as ethylene oxide, onto an aliphatic hydrocarbon corestructure bearing at least two nucleophilic groups (e.g., thiol, aminoor hydroxyl groups). Preferably, the nucleophilic groups are identicalsuch as in propane substituted with hydroxyl groups at the 1- and3-positions (1,3-propanediol) to, for example, favor polymerizationrates that are comparable in each of the polymer arms. At least onereactive group suitable for further modification, typically in protectedform such as a protected hydroxyl group, is also attached to thealiphatic hydrocarbon core, optionally via an intervening linkage.Following polymerization of the alkylene oxide monomer units onto thecore molecule, and optional end-capping of the poly(alkylene glycol)polymer arms, the protecting group of the protected hydroxyl or otherfunctional group is removed to provide a reactive group suitable forfurther modification, e.g., to form a branched polymer suitable fordirect covalent attachment to a biologically active molecule to form abranched polymer conjugate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter. Thisinvention may, however, be embodied in many different forms and shouldnot be construed as limited to the embodiments set forth herein; rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the invention to thoseskilled in the art.

I. DEFINITIONS

The following terms as used herein have the meanings indicated.

As used in the specification, and in the appended claims, the singularforms “a”, “an”, “the”, include plural referents unless the contextclearly dictates otherwise.

The terms “functional group”, “active moiety”, “reactive site”,“chemically reactive group” and “chemically reactive moiety” are used inthe art and herein to refer to distinct, definable portions or units ofa molecule. The terms are somewhat synonymous in the chemical arts andare used herein to indicate the portions of molecules that perform somefunction or activity and are reactive with other molecules. The term“active,” when used in conjunction with a functional group, is intendedto include those functional groups that react readily with electrophilicor nucleophilic groups on other molecules, in contrast to those groupsthat require strong catalysts or highly impractical reaction conditionsin order to react (i.e., “non-reactive” or “inert” groups). For example,as would be understood in the art, the term “active ester” would includethose esters that react readily with nucleophilic groups such as amines.Exemplary active esters include N-hydroxysuccinimidyl esters or1-benzotriazolyl esters. Typically, an active ester will react with anamine in aqueous medium in a matter of minutes, whereas certain esters,such as methyl or ethyl esters, require a strong catalyst in order toreact with a nucleophilic group. As used herein, the term “functionalgroup” includes protected functional groups.

The term “protected functional group” or “protecting group” or“protective group” refers to the presence of a moiety (i.e., theprotecting group) that prevents or blocks reaction of a particularchemically reactive functional group in a molecule under certainreaction conditions. The protecting group will vary depending upon thetype of chemically reactive group being protected as well as thereaction conditions to be employed and the presence of additionalreactive or protecting groups in the molecule, if any. Protecting groupsknown in the art can be found in Greene, T. W., et al., PROTECTIVEGROUPS IN ORGANIC SYNTHESIS, 3rd ed., John Wiley & Sons, New York, N.Y.(1999).

The term “linkage” or “linker” (L) is used herein to refer to an atom ora collection of atoms used to link, preferably by one or more covalentbonds, interconnecting moieties such as two polymer segments or aterminus of a polymer and a reactive functional group present on abioactive agent. A linker of the invention may be hydrolytically stableor may include a physiologically hydrolyzable or enzymaticallydegradable linkage.

A “physiologically hydrolyzable” or “hydrolytically degradable” bond isa weak bond that reacts with water (i.e., is hydrolyzed) underphysiological conditions. Preferred are bonds that have a hydrolysishalf life at pH 8, 25° C. of less than about 30 minutes. The tendency ofa bond to hydrolyze in water will depend not only on the general type oflinkage connecting two central atoms but also on the substituentsattached to these central atoms. Appropriate hydrolytically unstable ordegradable linkages include but are not limited to carboxylate ester,phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether,imines, orthoesters, peptides and oligonucleotides.

A “hydrolytically stable” linkage or bond refers to a chemical bond,typically a covalent bond, that is substantially stable in water, thatis to say, does not undergo hydrolysis under physiological conditions toany appreciable extent over an extended period of time. Examples ofhydrolytically stable linkages include but are not limited to thefollowing: carbon-carbon bonds (e.g., in aliphatic chains), ethers,amides, urethanes, and the like. Generally, a hydrolytically stablelinkage is one that exhibits a rate of hydrolysis of less than about1-2% per day under physiological conditions. Hydrolysis rates ofrepresentative chemical bonds can be found in most standard chemistrytextbooks.

An “enzymatically unstable” or degradable linkage is a linkage that canbe degraded by one or more enzymes.

The term “polymer backbone” refers to the covalently bonded chain ofrepeating monomer units that form the polymer. The terms polymer andpolymer backbone are used herein interchangeably. For example, thepolymer backbone of PEG is —CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂ where ntypically ranges from about 2 to about 4000. As would be understood, thepolymer backbone may be covalently attached to terminal functionalgroups or pendant functionalized side chains spaced along the polymerbackbone.

The term “reactive polymer” refers to a polymer bearing at least onereactive functional group.

Unless otherwise noted, molecular weight is expressed herein as numberaverage molecular weight (M_(n)), which is defined as

$\frac{\sum\limits_{\mspace{20mu}}^{\;}{NiMi}}{\sum\limits_{\mspace{11mu}}^{\;}{Ni}},$wherein Ni is the number of polymer molecules (or the number of moles ofthose molecules) having molecular weight Mi.

The term “alkyl”, “alkenyl”, “alkynyl” and “alkylene” refers tohydrocarbon chains typically ranging from about 1 to about 12 carbonatoms in length, preferably 1 to about 6 atoms, and includes straightand branched chains. Unless otherwise noted, the preferred embodiment ofany alkyl or alkylene referred to herein is C1-C6alkyl (e.g., methyl orethyl).

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonchain, including bridged, fused, or spiro cyclic compounds, preferablycomprising 3 to about 12 carbon atoms, more preferably 3 to about 8.

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Multiple aryl rings may be fused, as in naphthyl or unfused, asin biphenyl. Aryl rings may also be fused or unfused with one or morecyclic hydrocarbon, heteroaryl, or heterocyclic rings.

“Heteroaryl” is an aryl group containing from one to four heteroatoms,preferably N, O, or S, or a combination thereof, which heteroaryl groupis optionally substituted at carbon or nitrogen atom(s) with C1-6 alkyl,—CF₃, phenyl, benzyl, or thienyl, or a carbon atom in the heteroarylgroup together with an oxygen atom form a carbonyl group, or whichheteroaryl group is optionally fused with a phenyl ring. Heteroarylrings may also be fused with one or more cyclic hydrocarbon,heterocyclic, aryl, or heteroaryl rings. Heteroaryl includes, but is notlimited to, 5-membered heteroaryls having one hetero atom (e.g.,thiophenes, pyrroles, furans); 5-membered heteroaryls having twoheteroatoms in 1,2 or 1,3 positions (e.g., oxazoles, pyrazoles,imidazoles, thiazoles, purines); 5-membered heteroaryls having threeheteroatoms (e.g., triazoles, thiadiazoles); 5-membered heteroarylshaving 3 heteroatoms; 6-membered heteroaryls with one heteroatom (e.g.,pyridine, quinoline, isoquinoline, phenanthrine,5,6-cycloheptenopyridine); 6-membered heteroaryls with two heteroatoms(e.g., pyridazines, cinnolines, phthalazines, pyrazines, pyrimidines,quinazolines); 6-membered heteroaryls with three heteroatoms (e.g.,1,3,5-triazine); and 6-membered heteroaryls with four heteroatoms.

“Heterocycle” or “heterocyclic” means one or more rings of 5-12 atoms,preferably 5-7 atoms, with or without unsaturation or aromatic characterand at least one ring atom which is not carbon. Preferred heteroatomsinclude sulfur, oxygen, and nitrogen. Multiple rings may be fused, as inquinoline or benzofuran.

“Heteroatom” means any non-carbon atom in a hydrocarbon analog compound.Examples include oxygen, sulfur, nitrogen, phosphorus, arsenic, silicon,selenium, tellurium, tin, and boron.

The term “drug”, “biologically active molecule”, “biologically activemoiety” or “biologically active agent”, when used herein means anysubstance which can affect any physical or biochemical properties of abiological organism, including but not limited to viruses, bacteria,fungi, plants, animals, and humans. In particular, as used herein,biologically active molecules include any substance intended fordiagnosis, cure mitigation, treatment, or prevention of disease inhumans or other animals, or to otherwise enhance physical or mentalwell-being of humans or animals. Examples of biologically activemolecules include, but are not limited to, peptides, proteins, enzymes,small molecule drugs, dyes, lipids, nucleosides, oligonucleotides,polynucleotides, nucleic acids, cells, viruses, liposomes,microparticles and micelles. Classes of biologically active agents thatare suitable for use with the invention include, but are not limited to,antibiotics, fungicides, anti-viral agents, anti-inflammatory agents,anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones,growth factors, steroidal agents, and the like.

“Polyolefinic alcohol” refers to a polymer comprising a polyolefinbackbone, such as polyethylene, having multiple pendant hydroxyl groupsattached to the polymer backbone. An exemplary polyolefinic alcohol ispolyvinyl alcohol.

As used herein, “non-peptidic” refers to a polymer backbonesubstantially free of peptide linkages. However, the polymer backbonemay include a minor number of peptide linkages spaced along the lengthof the backbone, such as, for example, no more than about 1 peptidelinkage per about 50 monomer units.

By “residue” is meant the portion of a molecule remaining after reactionwith one or more molecules. For example, a biologically active moleculeresidue in a branched polymer conjugate of the invention is the portionof a biologically active molecule remaining following covalent linkageto a branched polymer of the invention.

“Oligomer” refers to short monomer chains comprising 2 to about 10monomer units, preferably 2 to about 5 monomer units.

The term “conjugate” is intended to refer to the entity formed as aresult of covalent attachment of a molecule, e.g., a biologically activemolecule, to a reactive polymer molecule, preferably a branched reactivepolymer of the invention.

“Monofunctional” in the context of a polymer of the invention refers toa polymer possessing a single reactive functional group.

“Bifunctional” in the context of a polymer of the invention refers to apolymer possessing two reactive functional groups which may be the sameor different.

“Multifunctional” in the context of a polymer of the invention means apolymer having 3 or more functional groups attached thereto, where thefunctional groups may be the same or different. Multifunctional polymersof the invention will typically comprise from about 3-100 functionalgroups, or from 3-50 functional groups, or from 3-25 functional groups,or from 3-15 functional groups, or from 3 to 10 functional groups, orwill contain 3, 4, 5, 6, 7, 8, 9 or 10 functional groups attached to thepolymer backbone.

II. BRANCHED REACTIVE POLYMERS

In one aspect, the present invention provides branched reactive polymerscomprising at least two polymer arms, such as PEG arms, attached to acentral core through heteroatom linkages such as ether linkages. Thecentral core molecule is an aliphatic hydrocarbon having a carbon chainlength of at least three carbon atoms (i.e., propane, butane, pentane,and the like). Since the branched polymers of the invention combine atleast two polymer arms in a single molecule, a polymer with sufficientmolecular weight to impart beneficial properties to a biologicallyactive molecule, such as increased water solubility, can be formed usingshorter, easier to prepare polymer chains. The branched polymers of theinvention are preferably monofunctional, meaning the polymer moleculecontains only a single reactive site for conjugation to a biologicallyactive molecule. Use of a monofunctional polymer eliminates thepossibility of crosslinking with a biologically active molecule, such asa protein, which can lead to loss of activity.

As described in greater detail below, for branched polymers of theinvention comprising poly(alkylene glycol) polymer arms, such as PEGarms, the branched polymers are advantageously synthesized bypolymerizing alkylene oxide monomer units, such as ethylene oxide units,directly onto an aliphatic hydrocarbon core molecule substituted withtwo or more nucleophilic groups (e.g., thiol, amino or hydroxyl groups).In this manner, expensive and time-consuming polymer purification stepsassociated with prior art methods are avoided.

Typically, the total number average molecular weight of the branchedreactive polymers of the invention will be about 500 to about 100,000daltons (Da), preferably about 5,000 to about 60,000 Da, most preferablyabout 8,000 to about 40,000 Da. Each polymer arm of the branched polymerwill typically have a molecular weight of about 250 Da to about 50,000Da, more preferably about 2,500 to about 30,000 Da, and most preferablyabout 4,000 to about 20,000 Da. Branched polymers having a total numberaverage molecular weight of about 500 Da, about 1,000 Da, about 2,000Da, about 4,000 Da, about 5,000 Da, about 8,000 Da, about 10,000 Da,about 12,000 Da, about 15,000 Da, about 20,000, about 25,000 Da, andabout 30,000 Da are particularly preferred.

A branched reactive polymer of the invention will typically comprise atleast two water-soluble and non-peptidic polymer arms, such aspoly(ethylene glycol) arms, covalently attached to an aliphatichydrocarbon core structure bearing a single functional group. Ageneralized structure of the branched reactive polymers of the inventionis shown below:Y—(X)p-R(—X′-POLY)_(q)  Formula I

wherein:

R is an aliphatic hydrocarbon having a length of at least three carbonatoms;

each POLY is a water soluble and non-peptidic polymer, such as PEG;

X′ is a heteroatom linkage, preferably —NH—, —O—, or —S—;

X is a linker;

p is 0 or 1;

q is 2 to about 10, preferably 2 to about 5 (e.g., 2, 3, 4, or 5); and

Y is a functional group.

The aliphatic hydrocarbon core, R, preferably comprises 3 to about 12carbon atoms, more preferably 3 to about 7 carbon atoms, most preferably3 to about 5 carbon atoms. Core structures of 3, 4, and 5 carbon atomsin length are particularly preferred. The aliphatic hydrocarbon can belinear or branched and may include one or more heteroatoms in thehydrocarbon chain. In a preferred embodiment, the polymer arms, POLY,and the functional group, Y, are each attached to different carbon atomsof the core molecule. For example, in a three-carbon core embodiment,the POLY polymer arms are preferably attached at the 1- and 3-positionand the Y functional group is preferably attached at the 2-position.

The branched polymers of the invention are preferably symmetrical,meaning the polymer arms are symmetrically located on the central core,R (e.g., at the 1- and 3-position of a three-carbon aliphatic core). Asymmetrical arrangement lends itself to the preferential formation ofonly one polymer product having polymer arms of approximately the samenumber of subunits, since the initiation of the polymerization processshould occur at approximately equal rates in equivalent arm positionsextending from a symmetrical core.

A. Polymer Arms

In general, the water soluble and non-peptidic polymer portion of thebranched polymer structure (i.e., POLY in Formula I above) should benon-toxic and biocompatible, meaning that the polymer is capable ofcoexistence with living tissues or organisms without causing harm. Whenreferring to a branched reactive polymer, it is to be understood thatthe polymer can be any of a number of water soluble and non-peptidicpolymers, such as those described herein as suitable for use in thepresent invention. Preferably, POLY as designated in Formula I above ispoly(ethylene glycol) (PEG). The term PEG includes poly(ethylene glycol)in any of a number of geometries or forms, including linear forms (e.g.,alkoxy PEG or bifunctional PEG), branched or multi-arm forms (e.g.,forked PEG or PEG attached to a polyol core), pendant PEG, or PEG withdegradable linkages therein, to be more fully described below. Preferredfor forming the branched polymers of the invention are linear polymerssuch as linear forms of PEG.

In its simplest form, PEG has the formula—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—  Formula II

wherein n is from about 5 to about 1,200, typically from about 50 toabout 700.

End-capped polymers, meaning polymers having at least one terminuscapped with a relatively inert group (e.g., an alkoxy group), can beused as a polymer of the invention. For example, methoxy-PEG-OH, or mPEGin brief, is a form of PEG wherein one terminus of the polymer is amethoxy group, while the other terminus is a hydroxyl group that issubject to ready chemical modification. The structure of MPEG is givenbelow.CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—OH  Formula III

wherein n is as described above.

Multi-armed or branched PEG molecules, such as those described in U.S.Pat. No. 5,932,462, which is incorporated by reference herein in itsentirety, although less preferred, can also be used as the PEG polymerin the branched reactive polymers of the invention. For example, anexemplary branched PEG polymer can have the structure:

wherein:

poly_(a) and poly_(b) are PEG backbones, such as methoxy poly(ethyleneglycol);

R″ is a nonreactive moiety, such as H, methyl or a PEG backbone; and

P and Q are nonreactive linkages. In a preferred embodiment, thebranched PEG polymer is methoxy poly(ethylene glycol) disubstitutedlysine. As would be appreciated by one of skill in the art, use ofbranched polymers as the POLY polymer arms in the branched reactivepolymers of the invention would result in a polymer having multiplebranching points within the molecule. Such polymers, if utilized toprepare the branched structures of the invention, are attached to thealiphatic core structures provided herein not by polymerization but bycovalent attachment.

Although less preferred due to its multifunctional nature, the PEGpolymer may alternatively comprise a forked PEG. Generally speaking, apolymer having a forked structure is characterized as having a polymerchain attached to two or more active agents via covalent linkagesextending from a hydrolytically stable branch point in the polymer. Anexample of a forked PEG is represented by -PEG-YCHZ₂, where Y is alinking group and each Z is an activated terminal group for covalentattachment to a biologically active agent. The Z group is linked to CHby a chain of atoms of defined length. International Application No.PCT/US99/05333, the contents of which are incorporated by referenceherein, discloses various forked PEG structures capable of use in thepresent invention. The chain of atoms linking the Z functional groups tothe branching carbon atom serve as a tethering group and may comprise,for example, an alkyl chain, ether linkage, ester linkage, amidelinkage, or combinations thereof. In this embodiment of the invention,the resulting branched polymer is multifunctional, i.e., having reactivesites suitable for attachment to a biologically active molecule not onlyextending from the aliphatic core, but also extending from the forkedpolymer arm(s). As in the above case, such forked polymers, if utilizedto prepare the branched structures of the invention, are attached to thealiphatic core structures provided herein not by polymerization buttypically by covalent attachment.

Again, although less favored due to its multifunctional nature, the PEGpolymer may comprise a pendant PEG molecule having reactive groups, suchas carboxyl, covalently attached along the length of the PEG backbonerather than at the end of the PEG chain. The pendant reactive groups canbe attached to the PEG backbone directly or through a linking moiety,such as an alkylene group.

In addition to the above-described forms of PEG, the polymer arms (POLY)can also be prepared with one or more weak or degradable linkages in thepolymer backbone, including any of the above described polymers. Forexample, PEG can be prepared with ester linkages in the polymer backbonethat are subject to hydrolysis. As shown below, this hydrolysis resultsin cleavage of the polymer into fragments of lower molecular weight:-PEG-CO₂-PEG-+H₂O→-PEG-CO₂H+HO-PEG-

Other hydrolytically degradable linkages, useful as a degradable linkagewithin a polymer backbone, include carbonate linkages; imine linkagesresulting, for example, from reaction of an amine and an aldehyde (see,e.g., Ouchi et al., Polymer Preprints, 38(1):582-3 (1997), which isincorporated herein by reference.); phosphate ester linkages formed, forexample, by reacting an alcohol with a phosphate group; hydrazonelinkages which are typically formed by reaction of a hydrazide and analdehyde; acetal linkages that are typically formed by reaction betweenan aldehyde and an alcohol; ortho ester linkages that are, for example,formed by reaction between a formate and an alcohol; peptide linkagesformed by an amine group, e.g., at an end of a polymer such as PEG, anda carboxyl group of a peptide; and oligonucleotide linkages formed by,for example, a phosphoramidite group, e.g., at the end of a polymer, anda 5′ hydroxyl group of an oligonucleotide.

In one instance, the polymer arms having one or more hydrolyzablelinkages contained therein are prepared in a two-step polymerizationprocess which includes an intermediate step for inclusion of the desiredhydrolyzable linkage. That is to say, polymerization of, e.g., ethyleneoxide subunits, onto the central core is carried out to a certaindesired chain length and the reactive polymer termini extending from thecentral core are then coupled to short polymer chains suitablyfunctionalized at one end to react with the hydroxyl groups of theintermediate polymer arms extending from the core to introduce thehydrolyzable linkage(s). Further polymerization of ethylene oxidesubunits onto the polymer arms, now containing one or more hydrolyzablelinkages, is then carried out to prepare polymer arms of a desired chainlength.

It is understood by those skilled in the art that the term poly(ethyleneglycol) or PEG represents or includes all the above forms of PEG.

Any of a variety of monofunctional, bifunctional or multifunctionalpolymers that are non-peptidic and water-soluble can also be used toform the branched polymers in accordance with the present invention. Thepolymer backbone can be linear, or can be in any of the above-describedforms (e.g., branched, forked, and the like). Examples of suitablepolymers include, but are not limited to, other poly(alkylene glycols),copolymers of ethylene glycol and propylene glycol, poly(olefinicalcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid),poly(acrylic acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline,poly(N-acryloylmorpholine), such as described in U.S. Pat. No.5,629,384, which is incorporated by reference herein in its entirety,and copolymers, terpolymers, and mixtures thereof.

Typically, the two or more polymer arms of a branched polymer of theinvention are the same. That is to say, most preferably, the polymerarms are each a poly(ethylene glycol) or each a polyolefinic alcohol,etc. Generally, not only are the polymer arms composed of the same typeof subunits, but they also have identical geometries and similarmolecular weights. That is to say, in a most preferred embodiment of theinvention, the polymer arms extending from the aliphatic core areidentical.

B. Linker (X)

The branched polymers of the invention optionally include a linkage(i.e., X in Formula I) that joins a branching carbon of the aliphatichydrocarbon central core molecule with the functional group, Y. Thestructure of the X linkage is typically determined by the structure ofthe aliphatic hydrocarbon core used to form the polymers of theinvention and has an overall length of from 1 to about 40 atoms,preferably 1 to about 10 atoms, and most preferably 1 to about 5 atoms.Preferred linkages include heteroatoms such as —O— or —S—, -alkylene-,—O-alkylene-O—, -alkylene-O-alkylene-, -aryl-O— (e.g., -phenylene-O—),—O-aryl- (e.g., —O-phenylene), (—O-alkylene-)_(m), and(-alkylene-O—)_(m), wherein m is 1-10, preferably 1-5 (e.g., 1, 2, 3, 4,or 5). The alkylene groups of the X linkage are preferablyC1-C6alkylene, more preferably C1-C3alkylene, including methylene andethylene.

In some instances, it may be advantageous to have a linker (i.e., X inFormula I) extending the point of covalent attachment of thebiologically active agent away from the central aliphatic core. In suchparticular embodiments of the invention, the terminus for activation andsubsequent attachment to an active agent is then at a primary ratherthan at a secondary carbon position, thereby increasing the ease ofsubsequent modifications due to the increased reactivity of a primarycarbon in nucleophilic displacement reactions.

C. Functional Group (Y)

The Y functional group can be any functional group suitable for reactionwith a functional group on a biologically active molecule or afunctional group that is a precursor thereof. Examples of suitablefunctional groups include hydroxyl, active ester (e.g.,N-hydroxysuccinimidyl ester and 1-benzotriazolyl ester), activecarbonate (e.g., N-hydroxysuccinimidyl carbonate, 1-benzotriazolylcarbonate, p-nitrophenyl carbonate), acetal, aldehyde having a carbonlength of 1 to 25 carbons (e.g., acetaldehyde, propionaldehyde, andbutyraldehyde), aldehyde hydrate, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, hydrazide, thiol, alkanoic acidshaving a carbon length (including the carbonyl carbon) of 1 to about 25carbon atoms (e.g., carboxylic acid, carboxymethyl, propanoic acid, andbutanoic acid), acid halide, isocyanate, isothiocyanate, maleimide,vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide,glyoxal, dione, mesylate, tosylate, and tresylate.

Exemplary functional groups are also described in the followingreferences, all of which are incorporated by reference herein:N-succinimidyl carbonate (see e.g., U.S. Pat. Nos. 5,281,698,5,468,478), amine (see, e.g., Buckmann et al. Makromol. Chem. 182:1379(1981), Zalipsky et al. Eur. Polym. J. 19:1177 (1983)), hydrazide (See,e.g., Andresz et al. Makromol. Chem. 179:301 (1978)), succinimidylpropionate and succinimidyl butanoate (see, e.g., Olson et al. inPoly(ethylene glycol) Chemistry & Biological Applications, pp 170-181,Harris & Zalipsky Eds., ACS, Washington, D.C., 1997; see also U.S. Pat.No. 5,672,662), succinimidyl succinate (See, e.g., Abuchowski et al.Cancer Biochem. Biophys. 7:175 (1984) and Joppich et al., Makromol.Chem. 180:1381 (1979), succinimidyl ester (see, e.g., U.S. Pat. No.4,670,417), benzotriazole carbonate (see, e.g., U.S. Pat. No.5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. J. Biochem.94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13:354 (1991),oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal. Biochem.131:25 (1983), Tondelli et al. J. Controlled Release 1:251 (1985)),p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl. Biochem.Biotech., 11:141 (1985); and Sartore et al., Appl. Biochem. Biotech.,27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym. Sci. Chem.Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No. 5,252,714),maleimide (see, e.g., Goodson et al. Bio/Technology 8:343 (1990), Romaniet al. in Chemistry of Peptides and Proteins 2.29 (1984)), and Kogan,Synthetic Comm. 22:2417 (1992)), orthopyridyl-disulfide (see, e.g.,Woghiren, et al. Bioconj. Chem. 4:314 (1993)), acrylol (see, e.g.,Sawhney et al., Macromolecules, 26:581 (1993)), vinylsulfone (see, e.g.,U.S. Pat. No. 5,900,461).

In one embodiment of the invention, the Y functional group is aprotected functional group, such as a protected hydroxyl group offormula —O-Gp, wherein Gp is a protecting group. The Gp protecting groupcan be any of various hydroxyl protecting groups known in the art, suchas benzyl or other alkylaryl groups (e.g., groups having the formula—CH₂—Ar, wherein Ar is any aryl group), acetal, and dihydropyranyl.Other suitable protecting groups are described in Greene, T. W., et al.,PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 3rd ed., John Wiley & Sons, NewYork, N.Y. (1999). As would be readily understood, the protecting group,Gp, can be readily displaced from the molecule to form a hydroxyl group,which can be further modified to form other functional groups usingtechniques known in the art.

D. Core Structures

The branched polymers of the invention are composed of an aliphatichydrocarbon-based core (i.e., R in Formula I above) having a length ofleast three carbon atoms, preferably from 3 to 7 carbon atoms. That isto say, a central core structure will typically contain at its core anumber of carbon atoms selected from the following: 3, 4, 5, 6, 7 ormore carbon atoms. Preferred are core structures containing 3, 5 or 7core carbons. Although the carbon atoms of the central core may havepolymer arms extending from any of the aforementioned carbons,preferably but not necessarily, the overall branched polymer issymmetrical. That is to say, for a three-carbon core, the polymer armspreferably extend from positions 1 and 3, with a site suitable forcovalent attachment to a biologically active molecule extending from thecentral carbon or the carbon at position 2. Similarly, for a five carboncore, the polymer arms extend from positions 1 and 5, with a sitesuitable for covalent attachment to a biologically active moleculeextending from position 3, or polymer arms extending from positions 2and 4, or, if a highly branched structure is desired, with polymer armsextending from each of positions 1, 2, 4, and 5. Exemplary three-carboncore structures possessing as the nucleophile an oxygen atom directlyattached to carbons 1 and 3 are provided in the Examples. These examplesdemonstrate synthetic approaches for building core structures having avariety of (X)p groups. Preferably, the nucleophiles (heteroatoms)attached to the central core are the same, e.g., all oxygen, allnitrogen, etc.

Although less preferred, suitable for use in forming the branchedpolymers of the invention are unsymmetrical core structures such asthose derived from 2-aminopentanedioic acid (glutamic acid),2-aminosuccinic acid (aspartic acid), and the like. In utilizing corestructures such as these, the terminal acid groups are typicallyactivated for coupling with a reactive polymer to form the branchedpolymer core. Alternatively, the carboxylic acid groups are reduced witha reducing agent to form the corresponding diol, which then possessessites suitable for building the polymer chains, for example, by acatalyzed reaction of the N-protected diol with an appropriate monomersubunit and subsequent polymerization thereof directly onto the centralcore.

E. Exemplary Branched Reactive Polymer Structures

More specific structural embodiments of the branched polymers of theinvention will now be described, all of which are intended to beencompassed by the structure of Formula I above. The specific structuresshown below are presented as exemplary structures only, and are notintended to limit the scope of the invention.

One embodiment of the invention having a three-carbon core has thestructure:

wherein POLY, X, p, and Y are described above.

In a preferred embodiment of Formula Ia, the branched polymer of theinvention has the structure:

wherein:

Z is a capping group or a functional group; and

POLY, X, p, and Y are described above.

The Z group is preferably a relatively inert capping group, such asalkoxy (e.g. methoxy or ethoxy), alkyl, benzyl, aryl, or aryloxy (e.g.benzyloxy). Alternatively, the Z group is a functional group capable ofreadily reacting with a functional group on a biologically activemolecule, such as any of the functional groups discussed above for the Yfunctional group.

In another embodiment of Formula Ia, each POLY is PEG end-capped withmethoxy as shown below:

In yet another embodiment of Formula Ia, the X linkage is absent asshown below:

wherein:

Z, POLY, and Y are defined above. Preferably, Z is methoxy and POLY isPEG.

In a further embodiment of Formula Ia, the X linkage is one of thespecific linkages shown below:

wherein:

X is —CH₂CH₂—O—CH₂CH₂— or —O—CH₂CH₂—; and

Z, POLY and Y are defined above. Preferably, Z is methoxy and POLY isPEG.

As described above, other heteroatoms, such as —NH— or —S— could be usedin place of the —O-linkages illustrated in Formulas Ia, Ia₁, Ia₂, Ia₃,and Ia₄ above.

F. Method of Forming Branched Reactive Polymers

The branched polymers of the invention are formed by attaching polymerarms to a heteroatom-substituted aliphatic hydrocarbon core moleculehaving at least three carbon atoms, such as propane, via heteroatomlinkages (e.g., —NH—, —O— or —S—). Although the polymer arms may beattached to the aliphatic hydrocarbon structure by simply reactingterminal functional groups on preformed purified polymers with reactivenucleophiles on the aliphatic hydrocarbon core without departing fromthe invention, for poly(alkylene glycol) polymers, it is preferable inmany respects to directly polymerize alkylene oxide monomer units, suchas ethylene oxide, propylene oxide, or butylene oxide subunits, onto analiphatic hydrocarbon core bearing at least two available hydroxylgroups (or other nucleophilic groups such as amino or thiol groups). Asillustrated in the Examples, alkylene oxide units can be polymerizedonto, for example, an alcohol molecule using a catalyzed reaction toform ether-linked polymer arms, preferably using base catalysis althoughother catalysts such as metal or acid catalysts could also be employed.By polymerizing the alkylene oxide directly onto a suitablyfunctionalized aliphatic hydrocarbon core structure, the branchedpolymer can be formed without first forming and purifying high molecularweight polymers, which is technically challenging, expensive, andtime-consuming.

The aliphatic hydrocarbon core molecule comprises two or more availablenucleophilic groups, such as hydroxyl groups, depending on the number ofpolymer arms to be attached to the core molecule. In one particularembodiment, the aliphatic hydrocarbon has two hydroxyl groups. The coremolecule also bears at least one protected functional group, such as aprotected hydroxyl group (i.e., —O-Gp, where Gp is described above).Preferably, the aliphatic hydrocarbon is 1,3-dihydroxy-2-substitutedpropane, wherein the protected hydroxyl group is attached at the2-position, optionally via an intervening linkage (i.e., X in Formula Iabove). The presence of the protecting group prevents polymerization atthat position, thereby ensuring that at least one side chain of thealiphatic hydrocarbon core will be available for further modification,for example, to a form suitable for covalent attachment to abiologically active molecule.

The generalized structure for the aliphatic hydrocarbon core molecule ofthe invention is shown below:Y′—(X)p-R(-Nu)_(q)  Formula V

wherein:

Y′ is a protected functional group, such as a protected hydroxyl group,wherein the presence of the protecting group prevents polymerization atthe Y′ position on the aliphatic core, R;

Nu is a nucleophile, such as amino, thiol or hydroxyl; and

R, X, p and q are defined above.

Unlike certain prior art applications that utilize a polyol or polyaminecore molecule to form a highly cross-linked hydrogel, the presentinvention utilizes a nucleophile-substituted aliphatic hydrocarbon coremolecule to form a branched polymer suitable for covalent coupling to abiologically active molecule.

The generalized structure for a preferred hydroxyl-substitutedthree-carbon aliphatic hydrocarbon core structure is shown below:

wherein:

X, p, and Gp are defined above.

Exemplary core structures of Formula Va include2-benzyloxy-1,3-propanediol, 2-benzyloxyethoxy-1,3-propanediol, and2-benzyloxyethoxyethyl-1,3-propanediol. The core structures of Formula Vare either commercially available (See Examples 1-2) or can be preparedfrom commercially available reagents (See Examples 3-4).

Base-initiated polymerization of ethylene oxide onto ahydroxyl-substituted aliphatic hydrocarbon of Formula Va results in abranched polymer of Formula Ia where Y is —O-Gp and POLY is -PEG-OH.Thereafter, in order to form a monofunctional branched polymer, theterminal hydroxyl groups of the PEG polymer chains are preferablyalkylated (e.g., methylated to form MPEG) by reaction with an alkylatingagent, such as methyl toluenesulfonate.

Following alkylation of the terminus of the PEG chains, the protectinggroup, Gp, can be displaced by hydrolysis or hydrogenolysis to produce ahydroxyl group. As would be understood, the hydroxyl group can then bemodified or converted to other reactive groups as desired, such as thereactive groups listed above for the Y moiety of Formula I.

III. BIOLOGICALLY ACTIVE CONJUGATES OF BRANCHED REACTIVE POLYMERS

The present invention also includes biologically active conjugatescomprising a biologically active molecule covalently attached to abranched polymer of the invention. As noted above, the branched polymersof the invention are preferably although not necessarily monofunctional(e.g., they may also be bifunctional or less preferablymultifunctional), and the biologically active agent is preferablyattached to the branched polymer via a linkage formed from reaction ofthe functional group on the branched polymer and a functional group onthe biologically active agent.

The specific linkage will depend on the structure of the functionalgroups utilized, and will typically be governed by the functional groupscontained in the biologically active molecule. For example, an amidelinkage can be formed by reaction of a branched polymer bearing acarboxylic acid group, or an active ester thereof, in the presence of acoupling agent, such as DCC, DMAP, or HOBT, with a biologically activeagent having an amine group. Alternatively, a sulfide linkage can beformed by reaction of a branched polymer bearing a thiol group with abiologically active agent bearing a hydroxyl group. In anotherembodiment, an amine linkage is formed by reaction of a branched polymerbearing an amino group with a biologically active molecule bearing ahydroxyl group. In yet another embodiment, a branched polymer bearing acarboxylic acid is reacted with a biologically active molecule bearing ahydroxyl group in the presence of a coupling agent to form an esterlinkage. The particular coupling chemistry employed will depend upon thestructure of the biologically active agent, the potential presence ofmultiple functional groups within the biologically active molecule, theneed for protection/deprotection steps, chemical stability of themolecule, and the like, and will be readily determined by one skilled inthe art. Illustrative linking chemistry useful for preparing thebranched polymer conjugates of the invention can be found, for example,in Wong, S. H., (1991), “Chemistry of Protein Conjugation andCrosslinking”, CRC Press, Boca Raton, Fla. and in Brinkley, M. (1992) “ABrief Survey of Methods for Preparing Protein Conjugates with Dyes,Haptens, and Crosslinking Reagents”, in Bioconjug. Chem., 3, 2013.

The linkage (i.e., L₁ in Formula VI below) can be hydrolyticallydegradable so that the biologically active agent is released intocirculation over time after administration to a patient. Exemplaryhydrolytically degradable linkages include carboxylate ester, phosphateester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines,orthoesters, peptides and oligonucleotides. If desired, a hydrolyticallystable linkage, such as amide, urethane (also known as carbamate),amine, thioether (also known as sulfide), and urea (also known ascarbamide) linkages, can also be used without departing from theinvention.

A generalized structure for a biologically active conjugate of theinvention comprising a branched polymer of Formula I can be representedas shown below:D-L₁-(X)p-R(—X′-POLY)_(q)  Formula VI

wherein:

D is a biologically active molecule, such as a peptide, protein, enzyme,small molecule drug, dye, lipid, nucleoside, nucleotide,oligonucleotide, polynucleotide, nucleic acid, polysaccharide, steroid,cell, virus, liposome, microparticle, micelle, fat, electrolyte and thelike;

L₁ is a linkage resulting from the reaction of the functional group ofthe branched polymer (i.e., Y in Formula I) and a functional group onthe biologically active molecule; and

POLY, X, X′, q and p are defined above.

In one preferred embodiment, the biologically active conjugate has thestructure:

wherein:

D, L₁, POLY, X, and p are defined above.

A biologically active agent for use in coupling to a branched polymer ofthe invention may be any one or more of the following. Suitable agentsmay be selected from, for example, hypnotics and sedatives, psychicenergizers, tranquilizers, respiratory drugs, anticonvulsants, musclerelaxants, antiparkinson agents (dopamine antagnonists), analgesics,anti-inflammatories, antianxiety drugs (anxiolytics), appetitesuppressants, antimigraine agents, muscle contractants, anti-infectives(antibiotics, antivirals, antifungals, vaccines) antiarthritics,antimalarials, antiemetics, anepileptics, bronchodilators, cytokines,growth factors, anti-cancer agents, antithrombotic agents,antihypertensives, cardiovascular drugs, antiarrhythmics, antioxicants,anti-asthma agents, hormonal agents including contraceptives,sympathomimetics, diuretics, lipid regulating agents, antiandrogenicagents, antiparasitics, anticoagulants, neoplastics, antineoplastics,hypoglycemics, nutritional agents and supplements, growth supplements,antienteritis agents, vaccines, antibodies, diagnostic agents, andcontrasting agents.

Examples of active agents suitable for use in covalent attachment to abranched polymer of the invention include, but are not limited to,calcitonin, erythropoietin (EPO), Factor VIII, Factor IX, ceredase,cerezyme, cyclosporin, granulocyte colony stimulating factor (GCSF),thrombopoietin (TPO), alpha-1 proteinase inhibitor, elcatonin,granulocyte macrophage colony stimulating factor (GMCSF), growthhormone, human growth hormone (HGH), growth hormone releasing hormone(GHRH), heparin, low molecular weight heparin (LMWH), interferon alpha,interferon beta, interferon gamma, interleukin-1 receptor,interleukin-2, interleukin-1 receptor antagonist, interleukin-3,interleukin-4, interleukin-6, luteinizing hormone releasing hormone(LHRH), factor IX insulin, pro-insulin, insulin analogues (e.g.,mono-acylated insulin as described in U.S. Pat. No. 5,922,675), amylin,C-peptide, somatostatin, somatostatin analogs including octreotide,vasopressin, follicle stimulating hormone (FSH), insulin-like growthfactor (IGF), insulintropin, macrophage colony stimulating factor(M-CSF), nerve growth factor (NGF), tissue growth factors, keratinocytegrowth factor (KGF), glial growth factor (GGF), tumor necrosis factor(TNF), endothelial growth factors, parathyroid hormone (PTH),glucagon-like peptide thymosin alpha 1, IIb/IIIa inhibitor, alpha-1antitrypsin, phosphodiesterase (PDE) compounds, VLA-4 inhibitors,bisphosphonates, respiratory syncytial virus antibody, cystic fibrosistransmembrane regulator (CFTR) gene, deoxyreibonuclease (Dnase),bactericidal/permeability increasing protein (BPI), anti-CMV antibody,13-cis retinoic acid, macrolides such as erythromycin, oleandomycin,troleandomycin, roxithromycin, clarithromycin, davercin, azithromycin,flurithromycin, dirithromycin, josamycin, spiromycin, midecamycin,leucomycin, miocamycin, rokitamycin, andazithromycin, and swinolide A;fluoroquinolones such as ciprofloxacin, ofloxacin, levofloxacin,trovafloxacin, alatrofloxacin, moxifloxicin, norfloxacin, enoxacin,grepafloxacin, gatifloxacin, lomefloxacin, sparfloxacin, temafloxacin,pefloxacin, amifloxacin, fleroxacin, tosufloxacin, prulifloxacin,irloxacin, pazufloxacin, clinafloxacin, and sitafloxacin,aminoglycosides such as gentamicin, netilmicin, paramecin, tobramycin,amikacin, kanamycin, neomycin, and streptomycin, vancomycin,teicoplanin, rampolanin, mideplanin, colistin, daptomycin, gramicidin,colistimethate, polymixins such as polymixin B, capreomycin, bacitracin,penems; penicillins including penicllinase-sensitive agents likepenicillin G, penicillin V, penicllinase-resistant agents likemethicillin, oxacillin, cloxacillin, dicloxacillin, floxacillin,nafcillin; gram negative microorganism active agents like ampicillin,amoxicillin, and hetacillin, cillin, and galampicillin; antipseudomonalpenicillins like carbenicillin, ticarcillin, azlocillin, mezlocillin,and piperacillin; cephalosporins like cefpodoxime, cefprozil, cefibuten,ceftizoxime, ceftriaxone, cephalothin, cephapirin, cephalexin,cephradrine, cefoxitin, cefamandole, cefazolin, cephaloridine, cefaclor,cefadroxil, cephaloglycin, cefuroxime, ceforanide, cefotaxime,cefatrizine, cephacetrile, cefepime, cefixime, cefonicid, cefoperazone,cefotetan, cefinetazole, ceftazidime, loracarbef, and moxalactam,monobactams like aztreonam; and carbapenems such as imipenem, meropenem,pentamidine isethiouate, albuterol sulfate, lidocaine, metaproterenolsulfate, beclomethasone diprepionate, triamcinolone acetamide,budesonide acetonide, fluticasone, ipratropium bromide, flunisolide,cromolyn sodium, ergotamine tartrate and where applicable, analogues,agonists, antagonists, inhibitors, and pharmaceutically acceptable saltforms of the above. In reference to peptides and proteins, the inventionis intended to encompass synthetic, native, glycosylated,unglycosylated, pegylated forms, and biologically active fragments andanalogs thereof.

IV. EXAMPLES

The following examples are given to illustrate the invention, but shouldnot be considered in limitation of the invention. For example, althoughPEG is used in the examples to illustrate the invention, other polymersthat are useful in the practice of the invention are encompassed by theinvention as discussed above.

All PEG reagents referred to in the appended examples are available fromShearwater Corporation of Huntsville, Ala. All other reagents arecommercially available. All ¹HNMR data was generated by a 300 or 400 MHzNMR spectrometer manufactured by Bruker.

Examples 1-2 illustrate a method of forming a branched reactive polymerof the invention using a commercially available hydroxyl-substitutedaliphatic hydrocarbon core molecule (2-benzyloxy-1,3-propanediol). InExample 1, the branched polymer is functionalized with an acetaldehydediethyl acetal. In Example 2, the branched polymer is functionalizedwith a succinimidyl ester of butyric acid. Examples 3-4 illustrate amethod of synthesizing two additional core molecules having interveninglinkages between the aliphatic hydrocarbon core molecule and theprotected hydroxyl side chain. Examples 5-6 illustrate PEGylation of anenzyme with a branched polymer of the invention to form a conjugate.

Example 1 Synthesis of 2-(1,3-di-mPEGoxy-2-propanoxy) acetaldehydediethyl acetal A. Synthesis of 1,3-diPEGoxy-2-benzyloxypropane [MWpoly(ethylene glycol) (PEG)=9 kda]]

A 500 ml round bottom flask was charged with 250 ml of freshlydistilled, dry THF containing 2-benzyloxy-1,3-propanediol (0.84 g, 4.59mmole). Potassium naphthalenide was added (0.28 M, 16.4 ml) withcontinuous stirring under an inert atmosphere. The flask was then cooledto 0° C. in an ice bath. Ethylene oxide (50.0 ml, 1.02 moles) was addedvia a cooled syringe. The reaction was allowed to warm to roomtemperature and was stirred for 72 hours. The reaction was quenched bythe addition of 5 ml of 0.2M acetic acid. The solvents were removed byrotary evaporation and the crude material redissolved in 100 ml ofmethylene chloride. The product was precipitated by the addition of 400ml of diethyl ether and collected by filtration. The product was driedunder vacuum.

Yield: 42 g. (93%). ¹H nmr (400 MHz DMSO-d₆), δ7.25-7.34, (m, 5H), 4.6(s, 2H), 3.2-3.8 (m, 826H).

B. Methylation of 1,3-diPEGoxy-2-benzyloxypropane

1,3-diPEGoxy-2-benzyloxypropane [MW poly(ethylene glycol) (PEG)=9 kDa](5.0 g, 0.55 mmoles) from Step A was placed in a two-necked round bottomflask and dissolved in 150 ml of toluene. The flask was fitted with aseptum and a Dean-Stark trap and the compound was azeotropically driedunder an inert atmosphere. The trap was replaced with a reflux condenserand the temperature of the flask was kept at 45° C. by placing the flaskin a constant temperature oil bath. Methyl toluenesulfonate (1.62 ml,5.4 mmoles) and 2.8 ml of potassium t-butoxide solution (1.0 M in THF)was added and the reaction stirred for 3 hours. Methyl toluenesulfonate(0.81 ml) and 1.4 ml of potassium t-butoxide solution were then addedand the reaction was stirred for an additional 3 hours. The flask wasremoved from the oil bath and cooled to room temperature. The solutionwas transferred to a single-necked round bottom flask and the solventwas removed by rotary evaporation. The residue was dissolved in 5 ml ofmethylene chloride and precipitated by the addition of 50 ml of diethylether. The product was collected by filtration and dried under vacuum.

Yield: 4.2 g. ¹H Nmr (400 MHz DMSO-d₆), δ 7.25-7.34 (m, 5H) 4.6 (s, 2H),3.3-3.8 (m, 826H), 3.24 (s, 6H).

C. Debenzylation of 1,3-di-mPEGoxy-2-benzyloxypropane

1,3-di-mPEGoxy-2-benzyloxypropane [MW poly(ethylene glycol) (PEG)=9 kDa](2.9 g, 0.32 mmoles) from Step B was dissolved in 100 ml ethanol.Pd(OH)₂/C (0.5 g) and cyclohexene (10 ml) was added and the mixture wasrefluxed for 4 hours. After cooling to room temperature, the mixture wasfiltered and the filtrate solvent removed by rotary evaporation. Thecrude residual material was dissolved in 5 ml of methylene chloride andprecipitated by the addition of 50 ml of diethyl ether. The product wascollected by filtration and dried under vacuum.

¹H Nmr (300 MHz, DMSO-d₆), δ 4.76 (d, 1H), 3.3-3.8 (m, 826H), 3.24 (s,6H).

D. Synthesis of 2-(1,3-di-mPEGoxy-2-propanoxy) acetaldehyde diethylacetal

To 2-hydroxy-1,3-di-mPEGoxypropane from Step C (M.W. 9000 Da, 4.5 g,0.0005 moles) in dioxane (250 ml) is added sodium hydroxide (0.20 g,0.005 moles) and chloroacetaldehyde diethyl acetal (0.38 g, 0.0025moles) and the mixture is refluxed 24 h with vigorous stirring. Thesolution is concentrated to about 150 ml, cooled, and filtered. Thefiltrate is evaporated to dryness, dissolved in 100 ml of water, andextracted with methylene chloride (3×75 ml). The combined extracts aredried over sodium sulfate, concentrated, and the product precipitated byaddition of 300 ml of ether. The precipitated product is collected byfiltration and dried under vacuum.

This reaction demonstrates conversion of the 2-benzyloxypropaneprotecting group to a protected form of an aldehyde (acetaldehydediethyl acetal) suitable for covalent coupling with amino groups on aprotein or other biologically active agent.

Example 2 Synthesis of 2-(1,3-di-mPEGoxy-2-propanoxy) succinimidylbutyrate (mPEG2-SBA) (20 kDa)

A. Methylation of 1,3-diPEGoxy-2-benzyloxypropane

20 g of 1,3-diPEGoxy-2-benzyloxypropane (20 kDa) prepared as describedin Step A of Example 1 and 0.01 g of BHT(2,6-Di-tert-butyl-4-methylphenol) were dissolved in 400 ml of toluene.The resulting solution was azeotropically dried by distillation underreduced pressure. The residue was redissolved in 700 mL of anhydroustoluene and 14 mL of potassium tert-butoxide solution (1.0 M solution intert-butanol) and 3.0 ml of methyl-toluene sulfonate were addedseparately. The reaction mixture was stirred overnight at 45° C. undernitrogen.

The insoluble material was filtered and filtrate was evaporated todryness under reduced pressure. The residue was dissolved in 700 ml ofdeionized water and saturated with NaCl. The pH of the solution wasadjusted to 7.5 and it was then extracted with dichloromethane (300ml×2). The extracted dichloromethane was dried over Na₂SO₄, filtered,evaporated and precipitated with Et₂O (500 mL). The methylated productwas collected by vacuum filtration and dried under vacuum overnight.

Yield: 18.5 g ¹H nmr (DMSO-d6): δ 7.33 ppm (mult. —OCH₂C₆ H ₅), δ 4.61ppm (s. —OCH ₂C₆H₅), δ 4.31 ppm (t, —OCH₂CH ₂OMs), 3.5 ppm (br. mult.,PEG), δ 3.24 ppm (s, CH ₃OPEG-).

B. Debenzylation of 1,3-di-mPEGoxy-2-benzyloxypropane

18.0 g of 1,3-di-mPEGoxy-2-benzyloxypropane (20 kDa) from Step A wasdissolved in 225 ml of 5 mM phosphate buffer (pH 7.2) and 1.13 g of 10%Pd on charcoal was added. The suspension was hydrogenated 20 hours under40 psi of hydrogen.

The suspension was filtered to remove catalyst and the filtrate wassaturated with NaCl and the pH of the solution was adjusted to 3.0. Thesolution was extracted with dichloromethane (300 ml×2) and the combinedextracts were dried over Na₂SO₄, filtered, evaporated and precipitatedwith Et₂O (500 mL). The product was collected by vacuum filtration anddried in vacuum overnight.

Yield: 13.2 g; ¹H nmr (DMSO-d6): δ 4.76 ppm (d. HO—CH—); δ3.5ppm (br.mult., PEG), δ 3.24 ppm (s, CH ₃OPEG-).

C. Synthesis of 2-(1,3-di-mPEGoxy-2-propanoxy) butyric acid

2.5 g of 2-hydroxy-1,3-di-mPEGoxypropane (20 kDa) from Step B wasdissolved in 30 ml of toluene and the resulting solution wasazeotropically dried by distillation under reduced pressure. The residuewas redissolved in 30 mL of anhydrous toluene and 1 mL of potassiumtert-butoxide solution (1.0 M solution in tert-butanol), 2.5 mg of BHT,and 0.25 g of1-(3-bromopropyl)-4-methyl-2,6,7-trioxabicyclo[2,2,2,]octane were added.The reaction mixture was stirred overnight at 65° C. under nitrogen.

The solvent was evaporated to dryness under reduced pressure, theresidue dried under vacuum for 2 hours, and finally redissolved in 60 mlof deionized water. The pH of the solution was adjusted to 2.0 with 10%H₃PO₄. After stirring at pH 2.0 for 15 min., the pH of the solution wasadjusted to 12.0 with 1.0 N NaOH and stirred at pH 12.0 for 2 hours. Thehydrolyzed solution was saturated with NaCl and the pH adjusted to 3.0with 10% H₃PO₄. The solution was extracted with dichloromethane (100ml×2) and the combined extracts were dried over Na₂SO₄, filtered,evaporated and precipitated with Et₂O (100 mL). The product wascollected by vacuum filtration and dried in vacuum overnight.

Yield: 2.3 g GPC: 79%.

D. Purification of 2-(1,3-di-mPEGoxy-2-propanoxy) butyric acid

The crude 2-(1,3-di-mPEGoxy-2-propanoxy) butyric acid from Step C waspurified by DEAE sepharose FF ion exchange column (100 mL). Afterpurification, the yield was 1.55 g.

¹H nmr (DMSO-d6): δ3.5 ppm (br. mult., PEG), δ 3.24 ppm (s, CH ₃OPEG-),δ 2.23 ppm (t, —OCH₂CH₂CH ₂COOH), δ 1.70 ppm (mult. —OCH₂CH ₂CH₂COOH).

E. Synthesis of 2-(1,3-di-mPEGoxy-2-propanoxy) succinimidyl butyrate (20kDa)

1.5 g 2-(1,3-di-mPEGoxy-2-propanoxy) butyric acid from Step D wasdissolved in 20 ml of anhydrous dichloromethane under nitrogen.N-hydroxysuccinimide (0.0132 g) was first added to the solution followedby 0.0234 g of dicyclohexylcarbodiimide. The solution was stirredovernight at room temperature under nitrogen. The product was filtered,concentrated under vacuum, precipitated into a mixture of IPA and Et₂O(1:1), collected by filtration and dried under vacuum.

Yield: 1.2 g; ¹H nmr (DMSO-d6): δ3.5 ppm (br. mult., PEG), δ 3.24 ppm(s, CH ₃OPEG-), δ 2.80 ppm (s, —NHS), δ 2.70 ppm (t. —OCH₂CH₂CH₂COONHS), δ 1.81 ppm (mult. —OCH₂CH ₂CH₂COONHS).

Example 3 Synthesis of (2′-benzyloxyethoxy)ethyl-1,3-propanediol(BEEP)—An Illustrative Aliphatic Hydrocarbon Core Molecule Suitable forUse in Preparing a Branched Polymer

A. Synthesis of di(ethylene glycol) monobenzyl ether methanesulfonate

Di(ethylene glycol) monobenzyl ether (15 g) in 200 ml of toluene wasdried by azeotropic distillation and the residue was redissolved in 400ml of anhydrous toluene and 100 ml of anhydrous dichloromethane. To thesolution was added 11.5 ml of dry triethylamine and 6.23 ml ofmethanesulfonyl chloride dropwise at 0-5° C. The reaction mixture wasstirred at room temperature under nitrogen overnight and the reactionwas quenched by adding 5 ml absolute ethanol. The insoluble material wasfiltered off and filtrate was evaporated to dryness. The residue wasredissolved in 200 ml of anhydrous toluene and insoluble material wasfiltered off. The filtrate was evaporated to dryness and residue wasdried under vacuum overnight.

Yield: 22 g ¹H nmr (DMSO-d6): δ 7.33 ppm (mult. —OCH₂C₆ H ₅), δ 4.49 ppm(s. —OCH ₂C₆H₅), δ 4.31 ppm (t, OCH₂ CH₂ OSO₂—CH₃), δ 3.69 ppm (t, OCH₂CH₂OSO₂—CH₃), δ 3.59 ppm (mult., —OCH₂CH₂ O—), δ 3.24 ppm (s,OCH₂CH₂OSO₂—CH₃ ).

B. Synthesis of C₆H₅—CH₂O—CH₂CH₂OCH₂CH₂—CH(COOC₂H₅)₂

Diethyl malonate (17.5 g) in 100 ml of 1,4-dioxane was added dropwiseNaH (3.6 g) in 150 ml of 1,4-dioxane under nitrogen. Di(ethylene glycol)monobenzyl ether methanesulfonate (10 g) from Step A in 600 ml of1,4-dioxane was added to the above diethyl malonate solution. Afterrefluxing the mixture for 4 hours, the reaction solution was filteredand evaporated to dryness. The residue was dried in vacuum overnight.

The remaining diethyl malonate was distilled off under reduced pressure.After distillation, the residue was purified by flash chromatography ona silica gel column eluted with hexane followed by dichloromethane. Thecombined dichloromethane extracts were evaporated to dryness and theproduct dried under vacuum overnight.

Yield: 6 g. ¹H nmr (DMSO-d6): δ 7.33 ppm (mult. —OCH₂C₆ H ₅), δ 4.48 ppm(s. —OCH ₂C₆H₅), δ 4.10 ppm (mult. OCH₂ CH₃), δ 3.51 ppm (mult.,—OCH₂CH₂ O—CH₂ CH₂—, —CH(CO₂—C₂H₅)₂), δ 2.01 ppm (mult. —OCH₂ CH₂—CH(CO₂—C₂H₅)₂), δ 1.16 ppm (t, —OCH₂ CH₃ ).

C. Synthesis of C₆H₅—CH₂O—CH₂CH₂OCH₂CH₂—CH(CH₂OH)₂

C₆H₅—CH₂O—CH₂CH₂OCH₂CH₂—CH(COOC₂H₅)₂ (5 g) from Step B was dissolved in200 ml of toluene and 29.5 ml of LiAlH₄ (1 M in THF) was added at 0-5°C. After stirring overnight at room temperature, 1 ml of water was addedfollowed by 1.0 ml of 15% NaOH and 3.0 ml of water. The insolublematerial was filtered and the filtrate was evaporated to dryness. Theproduct was purified by flash chromatography on a silica gel columneluted with ethyl acetate. Combined fractions were evaporated todryness. The final product was dried under vacuum overnight.

Yield: 1.5 g ¹H nmr (CDCl₃): δ 7.29 ppm (mult. —OCH₂C₆ H ₅), δ 4.55 ppm(s. —OCH ₂C₆H₅), δ 3.61 ppm (mult., C₆H₅CH₂—OCH₂CH₂ O—CH₂ CH₂—, —CH(CH₂OH)₂, δ 1.81 ppm (mult. —OCH₂CH₂—CH(CH₂OH)₂), δ1.65 ppm (mult. —OCH₂ CH₂—CH(CH₂OH)₂).

Example 4 Synthesis of (2′-benzyloxyethoxy)-1,3-propanediol—An ExemplarAliphatic Hydrocarbon Core Molecule Useful for Preparing a BranchedPolymer

A mixture of 2 g of cis-1,3-O-benzylideneglycerol, 3.51 ml of benzyl2-bromoethyl ether, 1.25 g of KOH powder and 30 ml of toluene wasstirred under reflux for about 20 hours. After cooling to roomtemperature, the insoluble material was removed by filtration and thefiltrate concentrated. The residue was distilled at 140° C. underreduced pressure to remove benzyl 2-bromoethyl ether. Afterdistillation, the residue dissolved in 20 ml of methanol containing 2 mlof conc. HCl and refluxed for 4 hours. 100 ml of water was added and thepH was adjusted to 5-6 with solid NaOH. NaCl was added to ˜10% and theproduct was extracted with dichloromethane (50 ml×3). The combineddichloromethane extracts were dried over Na₂SO₄, filtered andevaporated. The residue was dried under vacuum and the product waspurified by chromatography on a silica gel column (80 g) eluted withethyl acetate. The combined fractions were evaporated and dried undervacuum.

Yield: ˜0.9 g ¹H nmr (DMSO-d6): δ 7.31 ppm (mult. —OCH₂C₆ H ₅), δ 4.48ppm (s. —OCH ₂C₆H₅), δ 4.43 ppm (s. br. —CH(CH₂OH)₂).), δ 3.67 ppm (t.,—OCH₂ CH ₂O— CH(CH₂OH)₂), δ 3.54 ppm (t., —OCH₂ CH₂O—CH(CH₂OH)₂), δ3.41ppm (mult. —OCH₂CH₂O—CH(CH ₂OH)₂), δ3.29 ppm (mult.—OCH₂CH₂O—CH(CH₂OH)₂).

Example 5 PEGylation of Lysozyme with a Branched PEG Polymer

Lysozyme (0.0021 g, Sigma)) was dissolved in 1 ml of 50 mM sodiumphosphate buffer (pH 7.5) in a 2 ml vial. MPEG2 (20 kDa)-SBA (0.006 g, 2fold molar excess to the lysozyme) from Example 2 was added and thereaction vial was shaken at room temperature for 18 h.

The MALDI-TOF spectrum of the crude reaction mixture showed lysozyme,PEG2 (20 kDa)-butanoic acid, and mono-, and di-pegylated lysozyme atmasses of 14,028 Da, 21,810 Da, 35,612 Da, and 57,783 Da, respectivelyto be present. SDS-PAGE (10% Tris-HCL gel) displayed six bandsindicating tetra-, tri-, di-, mono-pegylated lysozyme (meaningpolymer-modified forms of the enzyme having 4, 3, 2, and 1 of thebranched polymers of the invention covalently attached thereto,respectively), PEG2 (20K)-butanoic acid, and unpegylated lysozyme.

Example 5 demonstrates the utility of the polymers of the invention informing conjugates having an amide linkage coupling the branched polymerstructure with a biological agent.

Example 6 PEGylation of Lysozyme with Branched PEG Polymer

2.2 mg, 1.9 mg, and 2.1 mg of lysozyme (Sigma) were dissolved in 1 ml of50 mM sodium phosphate buffer of pH 5.5, 6.5, and 7.6, respectively. 1.5mg of di-mPEG 2 kDa-butyraldehyde (5 fold molar excess relative to thelysozyme) and 0.1 mg of NaCNBH₃ (10 fold molar excess relative to thelysozyme) were added to the lysozyme solution of pH 5.5. 1.3 mg and 1.5mg of MPEG 2 kDa-butyraldehyde were added to the lysozyme solution of pH6.5 and 7.5, respectively, followed by the addition of 0.08 mg and 0.09mg of NaCNBH₃, respectively. The three reaction vials were shaken atroom temperature for 6 h.

Samples from the reactions of pH 5.5 and 6.5 showed the presence oflysozyme, mono-, and di-pegylated lysozyme by MALDI-TOF. Samples fromthe reaction conducted at pH 7.5 indicated the presence of onlydi-pegylated lysozyme by MALDI-TOF. After 24 h all reaction productmixtures contained lysozyme, mono-, and di-pegylated lysozyme, and,tri-pegylated lysozyme.

Three samples withdrawn from the pH 5.5, 6.5, and 7.5 reactions after 6hours were spotted on a 15% Tris-HCl gel. Each sample showed threevisible bands corresponding to di-, mono-, and native lysozyme. Threesamples withdrawn from the reactions of pH 5.5, 6.5, and 7.5 after 24hours showed four visible bands, which indicated tri-, di-, mono-, andunpegylated lysozyme.

Example 6 demonstrates the utility of the polymers of the invention informing conjugates wherein a biologically active agent is covalentlycoupled to the branched polymer via a secondary amine linkage generatedby reductive amination of the corresponding Schiff base.

1. A biologically active conjugate comprising a branched polymercovalently attached to a biologically active molecule, wherein theconjugate has the structure:D-L₁-(X)p-R(—O-POLY)_(q) wherein: D is the biologically active molecule;L₁ is a linkage resulting from the reaction of a functional group on thelinker (X), when present, or on the aliphatic hydrocarbon having alength of at least three carbon atoms (R) of the branched polymer and afunctional group of the biologically active molecule; R is an aliphatichydrocarbon having a length of at least three carbon atoms; each POLY isa water soluble and non-peptidic polymer that terminates with a hydroxylor methoxy group; X is a linker of 1 to 10 atoms in length; p is 0 or 1;and q is 2 to about 10, and further wherein the branched polymer has amolecular weight of about 12,000 Da to about 100,000 Da.
 2. Thebiologically active conjugate of claim 1, wherein D is selected from thegroup consisting of peptides, proteins, enzymes, drugs, dyes, lipids,nucleosides, nucleotides, oligonucleotides, polynucleotides, nucleicacids, polysaccharides, steroids, cells, viruses, liposomes, micelles,and fats.
 3. The biologically active conjugate of claim 1, having thestructure:


4. The biologically active conjugate of claim 1, wherein the branchedpolymer has a molecular weight of about 20,000 Da.
 5. The biologicallyactive conjugate of claim 1, wherein the branched polymer has amolecular weight of about 40,000 Da.
 6. The biologically activeconjugate of claim 1, wherein the branched polymer has a molecularweight of about 60,000 Da.